Primer Check? - (Jul/17/2012 )
Hi! I will try to keep this as short as possible:
I am trying to clone out the eGFP from this vector http://www.synthesisgene.com/vector/pEGFP-N3.pdf and replacing it with the mCerulean (sequence here: http://www.einstein.yu.edu/uploadedFiles/Research/Facilities/Fluorescent/spotlight/mCerulean%20revFinished.pdf) that I PCR out from a C1 vector.
So because it is an N3 vector, and the fluorophore is on the back, these primers would be appropriate to clone out the mCerulean:
Forward: GATC ggatcc A ATG GTG AGC AAG GGC GAG (Added the one A bp to put in frame from N3 to N1 position)
Reverse: GATC tctaga TTA CTT GTA CAG CTC GTC CAT (Added nothing)
Or, should I add two addition base pairs to the forward primer because it is coming out of a C1.
Any help is extremely appreciated!!
I checked it. You are digesting with BamHI and XbaI. you don't need the A nt in your forward primer. why did you add? you are amplifying mCerulean out and then insert it between BamHI and XbaI. you don't even need additional nts in your reverse. XbaI 1408 is right before SV40 and BamHI is in MCS. this replacement won't disturb any translation. just make sure your insert is in frame with mCerulean.
I also checked that BamHI and XbaI don't exist in your mCerulean. So go ahead without the additional A.
Thank you so much for your reply
I added the extra A to the mCer primer because I was changing it from a C1 vector to an N3 vector. I guess I was just confused! Thanks!!!